Pharmaceutical formulations commonly contain polysorbates 20 and 80 (PS20 and PS80), non-ionic surfactants composed of a hydrophilic polyoxyethylene head group and a hydrophobic fatty acid tail. The addition of surfactants to formulations protects proteins from surface-induced denaturation and aggregation (Geisen, Diabetologia 27:212-218 (1984); Wang, Int. J. Pharm. 289:1-30 (2005)). Protein aggregation can occur during drug substance (DS) and drug product (DP) processing, long-term storage, shipment, and during administration (Cromwell et al., AAPS J. 8:E572-E579 (2006)). It has been shown that the addition of a surfactant (e.g., PS20) can minimize interfacial interactions that may stress proteins during filtration (Maa et al., J. Pharm. Sci. 87:808-812 (1998); Maa et al., Biotechnol. Bioeng. 50:319-328 (1996)), agitation (Liu et al., J. Pharm. Sci. 102:2460-2470 (2013)), freeze-thaw (Kreilgaard et al., J. Pharm. Sci. 87:1597-1603 (1998); Hillgren et al., Int. J. Pharm. 237:57-69 (2002)), lyophilization (Carpenter, Protein Sci. 13:54-54 (2004); Carpenter et al., Pharm. Res. 14:969-975 (1997)), reconstitution (Webb et al., J. Pharm. Sci. 91:543-558 (2002)), administration (Kumru et al., J. Pharm. Sci. 101:3636-3650 (2012)), and storage.
To ensure stabilization of active pharmaceutical ingredients (API) during processing, long-term storage, and during administration, it is important to prevent polysorbate degradation. However, PS20 is susceptible to degradation via hydrolytic and oxidative pathways (Kumru, et al., J. Pharm. Sci. 101:3636-3650 (2012); Mahler et al., Abstr Pap Am Chem S. 239: (2010)).
Oxidative degradation of polysorbates has been well characterized and has been studied extensively (Kerwin, J. Pharm. Sci. 97:2924-2935 (2008); Kishore et al., J. Pharm. Sci. 100:721-731 (2011)). Oxidation typically occurs in the context of two mechanisms (1) the autoxidation of the ethylene oxide group and (2) radical oxidation at the site of unsaturation (Kishore et al., J. Pharm. Sci. 100:721-731 (2011)). Although oxidative degradation of polysorbates has been observed, it has been shown that PS20 oxidation can be mitigated in protein formulations by coformulating with antioxidants (e.g., methionine). Formulations containing tryptophan have also been developed to prevent oxidation of amino acid residues (US2014/0322203; US2014/0314) Oxidative and hydrolytic polysorbate degradation pathways are distinguishable by unique degradation product profiles. Hydrolytic polysorbate degradation produces predominantly fatty acids and oxidative polysorbate degradation produces more diverse degradation products including peroxides, aldehydes, acids, keytones, n-alkanes, fatty acid esters, and other degradation products (Ravuri et al., Pharm. Res. 28:1194-1210 (2011)).
Stress models for oxidative polysorbate degradation using 2,2′-Azobisisobutyramidinium (AAPH) that degrade PS20 have been described previously (Borisov et al., J. Pharm. Sci. 104:1005-1018 (2015)). Using similar approaches, representative stress models can be used to develop formulations that reduce oxidative polysorbate degradation under relevant conditions.
Stress models for hydrolysis using purified esterases (e.g., Porcine Liver Esterase, etc.) and lipases (e.g., tweenase, etc.) have been described previously (Labrenz, J. Pharm. Sci. 103L2268-2277 (2014)). Using similar approaches, representative stress models can be used to develop formulations that reduce catalytic polysorbate degradation under relevant conditions.
Recently, there have been reports of enzymatic degradation of polysorbate in monoclonal antibody (mAb) formulations. For example, Labrenz attributed polysorbate 80 (PS80) degradation observed in CHO-derived mAb formulations to specific enzymatic mechanism rather than a general biologic hydrolysis mechanism based on the PS20 degradation profile (Labrenz et al., J. Pharm. Sci. 103:2268-2277 (2014)). Sequencing of the CHO cell genome has identified various host cell proteins (HCPs) (e.g., lipases) capable of degrading polysorbate (S. Hammond et al., Biotech. Bioeng. 109:1353-1356 (2012)). Subsequently, Lee et al. have shown that reducing the expression of specific HCPs substantially reduced the hydrolysis of PS80 relative to control samples. These recent findings establish that lipases associated with biologics manufacturing are expressed in upstream processes. Downstream purification processes (e.g., Protein A) are capable of removal of HCPs; however, it has been shown that some HCPs can be co-purified with API molecules that have similar properties and are thus retained in trace quantities in the drug substance and drug product (K. Lee, et al., A Chinese Hamster Ovary Cell Host Cell Protein That Impacts PS-80 Degradation. AccBio Conference (2015). Presumably, lipases with high activity can result in significant polysorbate degradation even at undetectable levels. There are numerous ongoing efforts to identify and remove lipases from protein drugs by engineering cells with reduced lipase expression and via downstream processing steps (e.g., chromatography). However, the enzymatic degradation of PS20 and PS80 remains a significant challenge in biopharmaceutical development and there have been no significant efforts reported to identify optimal formulations for reducing hydrolytic or catalytic PS20 degradation.
Polysorbate degradation has numerous consequences that may impact the stability and shelf-life of protein drug formulations. Polysorbate degradants include poorly soluble fatty acids that may result in the formation of visible and subvisible particles in the solution. The loss of PS20 may also reduce the protective effects of PS20 for protein formulations. Additionally, a spiking study demonstrated that some of the PS20-related degradants can impact stability of protein drugs; however, no impact was observed under pharmaceutically relevant conditions (Kishore et al., Pharm. Res. 28:1194-1210 (2011).
What is needed is a method of reducing polysorbate degradation so that the protective effects of polysorbate on formulations (e.g., polypeptides) are maintained over time. This will result in more stable polypeptide formulations during processing, long-term storage, and during administration which in turn will lengthen the shelf life of polypeptide formulations and reduce waste caused by degraded and expired formulations.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.